A novel duplex qPCR-HRMA technique for simultaneous detection of Listeria monocytogenes and Salmonella typhimurium in meat products

Developing rapid, accurate, and sensitive methods to detect bacterial pathogens such as Listeria monocytogenes and Salmonella typhimurium is very important, given the global rise in foodborne outbreaks. To address this, we developed a duplex real-time PCR assay with high-resolution melting analysis (qPCR-HRMA) to detect these pathogens in meat products. The assay was standardized and validated according to ISO 22118:2011. The assay was optimized for basic PCR parameters and melting rate for HRM analysis. The reaction sensitivity was determined to be 2 pg of DNA, equivalent to 124 copies for Listeria monocytogenes and 100 copies for Salmonella typhimurium. The method sensitivity was found to be 150 CFU/mL for both pathogens in spiked meat samples. The assay was validated with proficiency test samples and was finally used to test real-world samples, where 4 samples were detected positive for the pathogens. This assay holds significant potential for regulatory food testing and clinical investigations.